No precise count has been made of how many soil specimens these workers have examined. Estimates put the figure at close to a million. As in the case of mineral prospectors on the old Western frontier, however, few of these modern hunters have made lucky strikes. The million or so samples analyzed to date have yielded fewer than 500 organisms secreting substances that are poisonous to other organisms. Fewer than a dozen of the 500 can be used in the treatment of human disease, and the secretions of only five are routinely prescribed by physicians. All the others are either too weak or too toxic to human beings or are effective only in the test tube. Yet in spite of its many disappointments the quest continues.

Last year the quest was joined by Bill Hulett, a high school student in Charleston, Miss. He reports that the thrill of rediscovering penicillin and two other known antibiotics more than rewarded his many failures. "Anyone who likes to combine the joys of the open country with the fascinations of the laboratory," Hulett writes, "should make a hobby of hunting antibiotics.

Soil organisms are fun to collect and interesting to grow. The analytical procedures are simple, inexpensive and full of surprises because the experimenter can never predict their outcome.

"My project began in 1963 when I came across a paper by the noted microbiologist Selman A. Waksman. In it he pointed out that although disease-producing organisms have been finding their way into the soil for millions of years, they have not crowded out all other organisms. Waksman suggested that, in the continuing struggle for survival, disease organisms in the soil must be destroyed by substances that are manufactured by other organisms.

"I wondered if such organisms inhabited our local soil. For background information I consulted my father, who is a physician, and borrowed some of his textbooks on bacteriology. Information on analytical procedures and culturing techniques was solicited from several manufacturers of antibiotics

"During this preliminary phase I learned that the destruction of microorganisms is not difficult. Even bacteria that produce the most deadly diseases in man can be killed by fairly mild treatment with heat, acid, alkali and other chemical compounds. The trick is to kill the offending microorganism without harming the human host. The most widely and frequently used drug for accomplishing this is penicillin, the antibiotic discovered in 1923 by Alexander Fleming. Unfortunately penicillin is not effective against all diseases; neither is any combination of the other widely used antibiotics, which include streptomycin, Chloromycetin, Aureomycin and Terramycin. I learned too that all groups of microorganisms must be regarded as potential sources of useful antibiotic drugs and that the amateur has as good a chance as anyone of discovering them.

"With that background and encouragement I set up my experiment. My soil specimens were collected from a variety of nearby locations: at the base of an oak tree, from a pine woods, a pecan grove, a sumac thicket and so on. The culture that produced the most organisms came from a meadow. The one that produced the fewest-one-was collected near my house. Yet the organism in this specimen secreted an antibiotic! The specimen that produced the most antibiotics-three-was collected near a fishpond. In all, antibiotic activity was found in six out of 23 specimens-a good batting average in spite of the fact that the six represented only three antibiotics, all of which are well known.

"The soil organisms were cultured on several media to encourage growth of the maximum variety. Organisms that grow as colonies were then isolated and subcultured in nutrient broth. The broth was tested for antibiotic activity by saturating a small disk of blotting paper with the solution and placing it on a test culture of bacteria. Activity was indicated by a circular zone surrounding the paper disk in which the growth of the bacteria was inhibited. The width of the zone varies in proportion to the strength of the antibiotic activity.

"For test bacteria I used Sarcina lutea, the harmless organism adopted by the National Bureau of Standards for the control of commercial penicillin production. It is easily grown and responds uniformly to the presence of antibiotics.. The zone of inhibition appears to the unaided eye as a clear circle surrounded by the bright yellow of the normal culture [Figure 5]

"To familiarize myself with the procedure I first made up a culture of Sarcina lutea and exposed it to several commercially prepared antibiotics. Small disks of blotting paper cut with a paper punch were moistened with drug solutions and placed on the plates of agar nutrient that had been freshly inoculated with Sarcina lutea. I had procured a culture of pure Penicillium notatum and cultivated it on nutrient broth under the same conditions I planned to use for culturing soil organisms. Blotting paper saturated with this broth was also tested on a nutrient agar plate of the Sarcina lutea. The resulting zones of inhibition varied from one to eight millimeters in width; the average was three millimeters.

"Some of the required apparatus, such as an incubator, can be constructed at home. I built an incubator of 1/4-inch plywood. It is essentially a box with a hinged door and an electric heater. The dimensions are not critical-any well-constructed cabinet of ample size will do. I joined the plywood rectangles by means of soft-pine corner strips that were fastened by brads and glue. The hinged door opened downward and was held shut by hooks or a magnetic latch of the kind used in kitchen cabinets. The latches, hinges, handles and other cabinet hardware were obtained from one of the large mail-order houses. The incubator was fitted with shelves cut from perforated aluminum of the do-it-yourself type stocked by most hardware dealers. One could substitute 1/4-inchsquare wire mesh of the kind known as hardware cloth if perforated aluminum were not available.

"The incubator was heated by five Christmas-tree lamp bulbs of Type No. 46. I installed miniature porcelain sockets for six bulbs but found that five heated the unit adequately. These lamps operate on 6.3 volts and are powered by the filament transformer of an old radio set. The heater assembly was installed in the bottom of the box, as shown in the accompanying illustration [right]. Room temperature did not vary widely, so that I did not equip the incubator with automatic temperature control. If automatic control were needed, it should be easy to improvise. One could substitute a single 60-watt lamp bulb for the miniature lamps and control it by a small thermostat of the kind used in chicken brooders. Such thermostats are listed in farm catalogues of the large mail-order houses for about $5.

"When it is used in locations where the temperature varies widely, the incubator should doubtless be insulated. Good insulation would be provided by an inch or so of rock wool in the space between a pair of nested boxes. Alternatively, the incubator could be made of slabs of foam plastic. My completed unit was sanded, painted with enamel and equipped with a door handle and a carrying handle.

"I also made several other pieces of apparatus: an alcohol lamp (used for sterilizing test tubes), inoculating needles, wire loops for manipulating solutions and a test-tube rack. The lamp consists of a small glass bottle, the neck of which makes a snug fit with a wick of sack cord 3/8 inch in diameter. I have also made lamps out of bottles with larger necks. These were fitted with a perforated cork that in turn fit the wicking. The cork was protected from the flame by a metal washer made of aluminum foil. Incidentally, holes of any desired size can easily be burned through corks by means of a red-hot file tine.

"My inoculation needles and wire loops were made of straightened paper clips. The end of each wire was inserted in a six-inch length of six-millimeter soft-glass tubing and the joint was heated in the flame of a Bunsen burner until the materials sealed. Wooden handles would have been as good but they were not as easy to make. A loop about an eighth of an inch in diameter was made at the outer end of some of the wires; this provided a handy device for picking up and transferring a large drop of culture solution.

"I was surprised at the number of shallow containers one requires for making these experiments. Initially I bought four Pyrex Petri dishes for 65 cents each. When the project got under way, it became apparent that I would need dozens of them, so I hunted for alternate vessels. Finally I found a source of pre-sterilized Petri dishes made of plastic. They are priced at $7.50 per 100 and are shipped in five sealed packages of 20 each. Eventually I used 250.

"Also required are three dozen test tubes approximately half an inch in diameter and five inches long. A rack for holding the test tubes can be made of hardware cloth. Mine measures six inches wide, eight inches long and four inches high. The mesh can be spread enough to accept the tubes. The top and ends of the rack consist of a single piece of mesh six inches wide and 16 inches long. Square bends made four inches from the ends serve as supports for the top. A second piece of mesh of the same width but only 10 inches long forms the bottom shelf of the holder. This nests inside the larger piece when square bends are made one inch from the ends [see illustration above]. Test tubes, stoppered by plugs of absorbent cotton, can be sterilized by being baked for 20 minutes at 300 degrees Fahrenheit in a kitchen oven.

"In general, I sterilized glassware in an autoclave-an old pressure cooker my mother had discarded. Six ounces of water was placed in the cooker along with the glassware and heated on the kitchen stove. A steam pressure of 15 pounds per square inch was maintained for 15 minutes to complete the sterilization. Because the pressure cooker was not large enough to take conventional half-liter flasks I used a variety of covered containers-peanut butter jars, fruit jars, jelly jars and so on-for agar preparations and other solutions.

"Harmless bacteria and penicillium cultures can be obtained from the American Type Culture Collection, 2112 M Street NW, Washington, D.C. 20036. (Pathogenic bacteria are not distributed to amateurs.) Substantial discounts are given on orders accompanied by documentary proof that the buyer is a student. Agar and related preparations can be procured from distributors such as Difco Laboratories, Detroit, Mich. 48201, and General Biological Supply House, 8200 South Hoyne Avenue, Chicago, Ill. 60620.

"In general, I maintained sterile conditions by means of techniques described in this department for March, 1958. I did not use a sterile transfer chamber, however, when manipulating the cultures. If Fleming had used a transfer chamber he might not have discovered penicillin, because the organism appeared on one of his cultures as a contamination. On the other hand, I did not deliberately encourage contamination. Only one culture was spoiled by what appeared to be an airborne organism.

"Five types of agar nutrient were tested initially. The first consisted of six grams of Difco nutrient agar added to 250 milliliters of water. Incidentally, distilled water was used for all preparations. The mixture was heated to approximately 150 degrees F. and stirred until the agar dissolved. It was then sterilized by autoclaving at a pressure of 15 pounds for 15 minutes.

"Agar medium of the second type consisted of 16 grams of BBL Sabouraud Dextrose agar; the third contained 14 grams of Difco Littman Oxgall agar. These nutrients were prepared in precisely the same way as the first type. The fourth type was specially compounded for the culture of actinomycetes. To 250 milliliters of distilled water I added five grams of Difco agar, .01 gram of ferrous sulfate, .1 gram of potassium chloride, .1 gram of magnesium sulfate, 2.5 grams of powdered skim milk and 2.5 milliliters of glycerine. The ingredients were heated to 150 degrees F., constantly stirred until dissolved and then autoclaved.

"The fifth type consisted of potato agar. Twenty-five grams of cubed potato was boiled for 10 minutes in 50 milliliters of water. The fluid was strained and to the clear solution was added 10 grams of glucose and eight grams of plain agar. Distilled water was then added to make a total volume of 250 milliliters, after which the preparation was autoclaved.

"The nutrient broth required for subculturing was prepared by boiling 10 grams of lean fresh chopped beef for 10 minutes in 50 milliliters of water. When it had cooled, the solution was filtered. Ten milliliters of unsulfured molasses, five grams of glucose and enough distilled water were then added to make up 250 milliliters. The resulting broth was autoclaved at a pressure of 15 pounds for 20 minutes.

"Each agar medium was poured while warm into previously sterilized plastic Petri dishes. On cooling, the material jelled. Approximately 20 milliliters was placed in each dish. Five milliliters of nutrient broth was placed in each test tube. All test tubes, after being plugged with a loose tuft of cotton, were then resterilized by autoclaving at 15 pounds for 20 minutes.

"Organisms were collected by digging about five inches below the surface of the ground and placing about 10 grams of soil in a sterile container with a close-fitting cover. Subsequently two grams of each specimen was transferred to a sterile test tube containing 10 milliliters of distilled water. The solution was shaken vigorously for 30 seconds and placed in the test-tube rack, where it was allowed to settle for five minutes. Subsequently a loopful of the clear fluid from the test tube was streaked over plates of agar.

"Early in the project tests disclosed that the maximum variety of organisms could be grown using only three of the five nutrients: nutrient agar, Sabouraud agar and Littman agar. Use of the actinomycetes agar and potato agar was discontinued. The organisms of every 4 soil specimen were thereafter cultured on agar plates of these three types.

"The inoculated plates were incubated at 84 to 88 degrees F. for intervals ranging from four to seven days, depending on apparent growth. All cultures were inspected daily. The colonies grow in varied forms. Some appear as irregular white patches, others as fuzzy greenish mounds. Still others seem to consist of white hair and to grow in concentric circles. Some are green with fuzzy white edges.

"As large, readily identifiable colonies appeared, specimens were transferred from the colony (by means of an inoculation needle) to nutrient broth for subculturing. The subcultures were incubated for intervals ranging from seven to 10 days at the same temperature as the primary cultures. Unfiltered broth was then tested for antibiotic activity. Incidentally, all culture vessels were labeled and detailed records were kept of every step in all experiments.

"For evaluating the activity of the subcultures, plates of nutrient agar were first inoculated with Sarcina lutea bacteria. A small disk of blotting paper was then cut with a paper punch from a sheet of paper that had been sterilized in the kitchen oven by being baked at 300 degrees F. for 30 minutes. The disk was moistened with the subcuiture broth to be tested, placed in the center of the inoculated agar plate and incubated at the same temperature as the cultures for seven days. Inhibition zones, if any, were then measured and recorded. In cases where antibiotic activity was indicated I immediately inoculated additional test tubes of nutrient broth with the responsible organism to ensure a continuing supply. The test was then repeated. In cases where plates became contaminated or a zone of inhibition was in doubt, the test was promptly repeated.

"In the course of the project I also checked a number of commercial antibiotics with the same procedures. SensiDisks bought from Difco Laboratories, for example, were used for observing the antibiotic activity of tetracycline, chloramphenicol, penicillin, triple sulfa, streptomycin and oxytetracycline. I also prepared penicillin by culturing Penicillium notatum strain No. 10002, obtained from the American Type Culture Collection. The organism was kept alive by weekly reculturing on potato agar and was tested by the same technique used for evaluating soil specimens. The resulting zones of inhibition were not as large as those produced by SensiDisks of penicillin but ranged from two to eight millimeters in diameter, averaging three millimeters.

"In all I analyzed 23 specimens of soil in four and a half months of experimenting. From these I identified 129 grossly different colonies of soil organisms and subcultured them in nutrient broth. Two of the specimens that exhibited antibiotic activity produced zones larger than three millimeters wide, two produced zones that ranged from one to three millimeters, and the zones of five measured less than one millimeter. Three cultures from the most interesting soil specimens were submitted to a commercial laboratory for evaluation. Two turned out to be penicillium molds. The third, which I had labeled specimen No. 113, proved to be a mixture of gram-positive and gramnegative bacteria. Only the gram-positive bacteria had antibiotic activity. This substance was identified as Fluvomycin, one of the lesser-known antibiotics, which is effective against pathogenic bacteria and fungi. As they say, you can t win em all-or even very many-but it is fun to try!"

Bibliography

MANUAL OF CLINICAL MYCOLOGY. Norman F. Conant, Donald Stover Martin, David Tillerson Smith, Roger Denio Baker and Jasper Lamar Callaway. W. B. Saunders Company, 1944.

The Society for Amateur Scientists (SAS) is a nonprofit research and educational organization dedicated to helping people enrich their lives by following their passion to take part in scientific adventures of all kinds.